1. Field of the Invention
This invention relates to methods for producing nucleotide sequences having regulatory functions using cellular selection of random nucleotide sequences, and to the sequences so produced.
2. Background Information
Every eukaryotic gene has a core promoter that resides at the extreme 5′ end of its transcription unit. Most core promoters contain common recognition sequences such as the TATA box and GC-rich motifs, which allow binding of RNA polymerase, the enzyme required for the synthesis of messenger RNA on DNA templates. The core promoter is essential for initiation of transcription. However, it alone usually does not contain all the information necessary for the modulated expression of a gene in different contexts in the developing or behaving organism. This contextual information is frequently provided by other regulatory elements such as enhancers and silencers, which reside in the gene at locations that are proximal to the core promoter either upstream or downstream from an initiation site of RNA transcription, and can be several kilobases away from the core promoter. In addition, the mRNA molecules transcribed from gene sequences contain translational regulatory elements, which regulate production of a polypeptide from the mRNA. For example, the mRNA can contain an internal ribosome entry site (IRES) sequence, which effects the manner in which ribosomes bind to an mRNA and initiate translation, and does not require interaction of the ribosome with the 5′ end of an mRNA transcript. Thus, an IRES element can confer an additional level of regulation on gene expression.
It is not completely understood how combinations of regulatory elements interact with the core promoter to achieve the remarkable contextual diversity of gene expression that exists during animal development and tissue regeneration, as well as the mis-regulation associated with pathological conditions such as neoplastic disorders. Understanding how this diversity comes about is a major goal of modern biology, and achievement of this goal would accelerate progress in a number of areas in cell biology, development, and medicine. For instance, synthetic promoters or IRESes that function in a tissue specific manner, and that are selected as markers of either healthy or diseased tissues, can be useful in diagnostic or therapeutic procedures, and in drug development. Such applications for these promoters also can extend our understanding of a variety of diseases, thus providing a means to develop therapeutic interventions.
Eukaryotic promoters are complex and frequently contain combinations of several transcriptional regulatory elements. These DNA motifs are recognized by specific proteins (transcription factors) that bind to the element and regulate transcription of a particular gene. Hundreds of DNA segments that participate in the regulation of transcription of genes in eukaryotic systems have been characterized. However, these elements and their corresponding transcription factors generally have been analyzed only as individual units, for example, as to how an element and its associated transcription factors regulate the expression of a particular gene in a specific context. However, the rules by which regulatory elements function either by themselves or in combination with other elements in the many genes in which these elements are found are not well understood.
An example of this complexity is provided by the specific interaction of activator protein 1 (AP-1) with the TPA responsive gene regulatory element (TRE), which is present in the promoter and enhancer regions of many eukaryotic genes. The TRE is bound by members of the fos and jun families of transcriptional regulatory proteins, which are recruited in a number of regulatory situations in gene expression, particularly under conditions involving the integration of growth factor signals. A TRE can be present in a regulatory region of a gene that is expressed only in the kidney during its differentiation or, alternatively, in a gene that is expressed constitutively by neural cell precursors. It is not known, however, how the element is selected to function in a very specific context in each of these different environments or, for example, whether other elements are involved in modulating the function of a TRE such as the ability to repress (or potentiate) activity from the TRE.
Compared to transcriptional control sequences, little is known about translational control sequences. Some IRESes have been identified in viruses, and more recently cellular mRNA sequences having IRES activity have been identified. Unlike transcriptional regulatory elements, however, small modular elements having translational regulatory activity, including IRES activity, have not been identified.
Currently, there is no general systematic framework for analyzing the anatomy of promoters, enhancers, IRESes and other transcriptional and translational regulatory elements, and it is unknown how the combination of several common transcriptional and translational motifs present in many of these regulatory elements function cooperatively to create unique patterns of gene expression. For example, particular variations of nucleotides within a regulatory element may be able to function well in the context of a specific companion element, while other variants of the motif may be able to override the influences of neighboring elements. Thus, a need exists for methods to identify functional transcriptional and translation regulatory elements. The present invention satisfies this need and provides additional advantages.